Hepatitis C virus (HCV) is a major public health concern and a leading cause of chronic liver disease and liver cancer. Standard HCV treatment options are limited, and understanding the virus-host interface regulating innate immunity against HCV is necessary to develop new therapies and restrict infection. Studies in our laboratory have revealed that HCV RNA is recognized as non-self by RIG-I, a cytoplasmic pathogen recognition receptor, through binding of the poly-U/UC domain located in the 3' non-translated region of the HCV RNA. In addition, our studies have demonstrated that the HCV NS3/4A protease mediates viral evasion of the host innate antiviral immune response through cleavage of the RIG-I binding protein IPS-1. Finally, substantial genetic diversity between HCV genotypes and quasi-species results in variable regulation of the RIG-I signaling pathway, presenting a major obstacle for innate and adaptive antiviral immune responses, and is a key factor supporting chronic infection and pathogenic outcome. Taken together, our studies connect the RIG-I signaling pathway and the HCV NS3/4A protease as a major virus-host interface regulating innate immunity and HCV infection outcome. The main goal of this proposal is to define the relationship between HCV genetic diversity and viral regulation of innate antivira immunity. In this proposal, we will investigate the hypothesis that HCV activation and suppression of the RIG-I signaling pathway regulates the innate immune response against infection, and variation within these processes due to HCV genotype and quasispecies diversity may affect the virologic and clinical correlates of infection. Specifically, we will (1) identify features of the HCV poly-U/UC motif and RIG-I that trigger innate immune signaling, (2) assess genetic and functional variation in the poly-U/UC motif among HCV genotypes and within the viral quasispecies, and (3) analyze HCV NS3/4A genetic diversity and determine the ability of NS3/4A from different HCV genotypes to cleave IPS-1 and block innate immune signaling. The proposed studies will reveal the role of HCV genetic diversity in the regulation of innate immunity, thus providing new insights for therapeutic and vaccine design strategies aimed at restricting HCV infection. PUBLIC HEALTH RELEVANCE: Hepatitis C virus (HCV) is a major public health concern and a leading cause of chronic liver disease and liver cancer worldwide. Approximately 170 million people worldwide are persistently infected with HCV, and standard treatment is currently limited to interferon-based therapies that result in viral clearance in only 50% of infected subjects. Vaccine strategies are not currently available for preventing HCV infection, so understanding the virus-host interactions regulating innate immunity against HCV is necessary in order to develop new therapies and restrict infection.